Project Summary: Cardiovascular disease (CVD) is the leading cause of morbidity and mortality worldwide. Heart failure (HF), a major form of CVD, results from cardiomyocyte (CM) hypertrophy and apoptosis, combined with cardiac fibroblast (CF) activation and proliferation. Although TGF-b-IL-11 axis induced protein synthesis of pro-fibrotic genes have been observed, the mechanisms that promote pro-fibrotic mRNA translation in CF have not been identified. Therefore, filling this gap and identifying the pharmacologically targetable translational control determinants of cardiac fibrosis is of paramount importance. Aminoacyl-tRNA synthetases (ARSs) catalyze the ligation of amino acids to cognate tRNAs. In mammals, glutamyl-prolyl-tRNA synthetase (EPRS) catalyzes the attachment of glutamic acid (E) and proline (P), to their cognate tRNAs for protein synthesis. By screening of ARSs induced in TGF-b- activated human CFs, human ARSs with SNPs in CHD, mouse ARSs associated with ISO-induced cardiomyopathy by GWAS, and ISO-induced ARSs in mouse failing hearts, we have identified EPRS as the key ARS involved in various cardiac pathologies. In this project, we uncovered EPRS as an integrated node downstream of multiple pro-fibrotic stimuli. We found that activating transcription factor 5 acts as an upstream transcriptional regulator of EPRS. Halofuginone (Halo), a Chinese herbal medicine-derived chemical compound, is a prolyl-tRNA synthetase-specific inhibitor. Low-dose Halo, as well as genetic knockout of one allele of EPRS in the mouse genome, reduces cardiac hypertrophy and fibrosis in multiple HF mouse models. We employed RNA-Seq and polysome profiling-Seq in Halo- treated fibroblasts to define gene expression changes at the transcriptomic and translatomic level upon EPRS inhibition. We identified novel proline codon rich (PRR) genes in addition to collagens, which are upregulated by EPRS at the posttranscriptional level and may play critical roles in cardiac fibrosis. Inactivation of EPRS promotes translational repression of PRR mRNA coupled with enhanced mRNA decay. This effect requires inactivation of eIF5A, an elongation factor for decoding Pro-rich codons. Our central hypothesis is: MI stress-induced EPRS promote cardiac fibrosis via increased Pro-tRNAPro pool, and enhanced stabilization and translational activation of pro-fibrotic PRR mRNAs in CFs. We will test this hypothesis by pursuing 3 aims. Aim 1. Determine if downregulation, globally and in CFs, and pharmacological inhibition of EPRS prevent or reverse cardiac fibrosis in HF models. Aim 2. Determine novel preferential downstream PRR pro-fibrotic mRNA targets of EPRS and upstream regulatory pathway of EPRS. Aim 3. Determine mechanisms of eIF5A-mediated translational control and mRNA decay of PRR genes. This project will promote the development of novel therapeutic approaches by inhibiting translation factors, and identification of novel pharmacological targets.